In studying structure-function relationships of iron proteins, spectroscopic techniques like EPR and Mossbauer have proved invaluable for understanding the chemical nature of the active center. The spectroscopic parameters provide a clear way of comparing one iron protein with others having different catalytic properties. The proposed project involves the physical characterization of heme model compounds and mutant forms of myoglobin. We are interested in heme model compounds having a) pi-cation radicals, b) a ferric "mixed-spin" iron state, or c) a low-spin ferric iron with an unusual dxy ground state. The cation radicals are models for intermediate states in the catalytic cycles of many mammalian heme enzymes, such as the peroxidases. Our studies on heme models will be done in collaboration with Dr. W. R. Scheidt. We hope to clarify how the peripheral constituents and axial ligands of the heme influence 1) the ground spin state of the iron, 2) the orbital occupied by the cation radical, and 3) the magnetic coupling between the iron and the radical. We will specifically use Mossbauer and EPR spectroscopy to study heme compounds related to the octaethyl porphyrin (OEP) model [Fe(OEP')C1]2(SbC16)2 and the equivalent compound having one more electron and a single counterion. The spectroscopic and magnetic parameters of the models will be determined by computer simulation, and will be correlated with structural information from x-ray diffraction measurements. The case for an iron protein being in the "mixed spin" state was first convincingly made for ferricytochrome c'. Since then, many other enzymes and model compounds have exhibited this state. We would like to clarify the chemical conditions necessary to stabilize this state. The low-spin ferric heme models of interest are those related to the tetraphenyl porphyrin (TPP) model [Fe(TPP)(CNBu)2]+ which, unlike nearly any other low-spin ferric system, has an axial low=spin EPR spectrum and may have its unpaired electron in a dxy orbital. We hope to elucidate the bonding properties required of the axial ligands in order to produce this unusual state. The myoglobin studies will be done in collaboration with Dr. S. G. Sligar, and will focus on clarifying how the properties of the iron ligand binding site are perturbed by modifications of the polypeptide backbone. Recombinant techniques will be used to generate mutations of the E and F chains. The mutant myoglobins will be investigated by looking at the binding properties of CO and O2 relative to the wild type, and by Mossbauer spectroscopy and EPR. The Mossbauer parameters will be analyzed in a crystal field model, which should afford insight into how crystal field potentials correlate with small structural changes in a protein, and how those structural changes in turn influence the reactivity of the heme binding site. To facilitate the analysis of the Mossbauer data on all of these systems, we will develop an iterative multiparameter computer fitting program based on modern minimization algorithms.